Project Summary/Abstract A fraction of germinal center-derived B cell non-Hodgkin lymphomas (~12% of Burkitt lymphomas, ~9% of Diffuse Large B cell lymphomas, up to 36% of relapsed Diffuse Large B cell lymphoma cases) carry recurrent missense mutations in FOXO1, a member of the Fox-O family of Forkhead transcription factors. FOXO1 controls critical stage transitions during B cell development, and is a main target of the PI3K/AKT pathway, which relays key survival signals downstream of the B cell receptor. While FOXO1 is inactivated and thus classically regarded as a tumor suppressor in diverse cancer types and in animal models, lymphoma- associated FOXO1 mutations are predicted instead to lock FOXO1 in a constitutively active state by rendering it insensitive to PI3K-dependent nuclear export and degradation. In a recent study we found that FOXO1 is critical for the maintenance of specific B cell fate programs required for the control of zonal polarity in the germinal center. Consistently, FOXO1 null germinal centers fail to support some key biological functions that are typically associated to zonal polarity, including affinity maturation and class-switch recombination. This project seeks to understand how lymphoma-associated mutations impact FOXO1 activities in germinal center B cells, and how these events may contribute to the pathogenesis of germinal center-derived B cell non-Hodgkin lymphomas. We propose that FOXO1 mutations actively contribute to B cell lymphomagenesis by altering the control of germinal center polarity and B cell responses to specific immune signals. To test this idea, we will develop the following specific aims: 1) Investigate how FOXO1 mutations alter B cell responses driven by stress-activated protein kinase pathways, combining in vitro and in vivo chemical genetics strategies in normal, cancerous germinal center B cells to measure and manipulate FOXO1 responses to these specific pathways; with further validation through the analysis of primary B-NHL cases; 2) Determine the impact of FOXO1 mutations on germinal center B-cell homeostasis, by engineering germinal center B cells with specific missense mutations in order to evaluate their effects on germinal center polarity and the control of germinal center-specific FOXO1 transcriptional programs; 3) Evaluate the role of FOXO1 mutations in the pathogenesis of MYC-driven B-cell lymphomas, by using adoptive transfer strategies to mimic in vivo the genetics of a fraction (~12%) of Burkitt lymphomas in which MYC deregulation and FOXO1 mutations coexist. Given the role of FOXO1 in normal germinal center physiology, the results of this study are expected to provide direct insights into cellular and molecular mechanisms involved in the pathogenesis of germinal center-derived B cell non-Hodgkin lymphomas. These studies are likely to be clinically relevant because of the growing interest on PI3K inhibitors as therapeutic agents in this group of malignancies.